Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 3 (march 2020) : 293-299

Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Manju Singh1, Saurabh Gupta2, Shoor Vir Singh2, Gururaj Kumaresan3, Deepansh Sharma1, G.K. Aseri1, Parul Yadav1, Rathnagiri Polavarapu4, Jagdip Singh Sohal1,*
1Amity Center for Mycobacterial Disease Research, Amity Institute of Microbial Technology, Amity University Rajasthan, Kant-Kalwar, Jaipur-303 002, Rajasthan, India.
2Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University, Mathura-281 406, Uttar Pradesh, India.
3Animal Health Division, Central Institute for Research on Goats, Farah, Mathura-281 122, Uttar Pradesh, India.
4Genomix Molecular Diagnostics Pvt. Ltd., Kukatpally, Hyderabad-500 072, Andhra Pradesh, India.
Cite article:- Singh Manju, Gupta Saurabh, Singh Vir Shoor, Kumaresan Gururaj, Sharma Deepansh, Aseri G.K., Yadav Parul, Polavarapu Rathnagiri, Sohal Singh Jagdip (2020). Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease . Indian Journal of Animal Research. 54(3): 293-299. doi: DOI: 10.18805/ijar.B-3775.

ABSTRACT

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP  for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

INTRODUCTION

Johne’s disease (JD or paratuberculosis) is one of the most lethal infectious chronic granulomatous enteritis prevalent in domestic and wild ruminants worldwide (Singh et al., 2014a). Disease has severe economic impact to dairy and meat industry due to the loss of milk production and early culling or death (Singh et al., 2018; Ahlstrom et al., 2015). MAP infects young livestock through ingestion of MAP contaminated milk. MAP has also been associated with certain human illnesses such as Inflammatory bowel disease (Crohn’s disease, Ulcerative colitis) and type 1 diabetes (Sechi et al., 2015). Human population is exposed to this pathogen by milk since MAP remains live even after pasteurization (Singh et al., 2018). Several studies have reported presence of MAP bacilli from commercially pasteurized milk and milk products (Singh et al., 2018).
       
Control and eradication practices for MAP consist of hygienic management, test and cull, restricting animal movements and vaccination (Singh et al., 2014b). Being the most common, acid fast staining is a simple and cheap test to diagnose the presence of MAP in feces suffers from low sensitivity and specificity (Hermel, 1998). Furthermore, early immuno-diagnostic testing was constrained to CMI based intradermal test (Johnin test); however this test also has poor specificity and sensitivity (Hermel, 1998). Lately, attempts have been made to recognize antibodies response to MAP infection by ELISA, yet available ELISA assay is based on whole cell antigen, in this way specificity is an issue because of cross reactive antigens from environmental mycobacteria (Chaubey et al., 2016). Fecal culture considered as ‘Gold standard’ also has drawbacks as it is time-consuming, labor-intensive, requiring atleast 8-12 weeks to grow and has low sensitivity, evaluated to be at 33.0% in feces (Wells et al., 2000). Fecal PCR is better alternative to culture as ‘Gold Standard’ test but investigations have demonstrated that fecal PCR has variable sensitivity due to inhibitory substances, low load of MAP bacilli, inefficient DNA yield (Wells et al., 2006).
       
Since, MAP has insidious nature, long incubation period and lack of early, rapid and accurate diagnostic tests therefore, it is difficult to control and eradicate JD in domestic livestock population (Singh et al., 2007). Therefore, to initiate a national level control program, development of improved diagnostic tools are required that can sensitively detect disease as early as possible so that infected animal can be segregated. To overcome the limitations of the available diagnostic tests this study was aimed to develop an visual LAMP assay for the detection of MAP infection in fecal samples of infected animals, which is user friendly, cost effective, simple and has high sensitivity and specificity (upto femtograms) over traditional PCR methods (upto nanograms).

MATERIALS AND METHODS

MAP reference strain
 
Mycobacterium avium subsp. paratuberculosis ‘S 5’ strain ‘Indian Bison Type’, Mycobacterium tuberculosis and Mycobacterium avium were procured from mycobacterial repository, Microbiology laboratory, CIRG, Makhdoom. To standardize the LAMP assay, reference ‘S 5’ strain of MAP was sub-cultured modified Herrold’s egg yolk medium (HEYM) supplemented with mycobactin J as per Singh et al., (1996). After 4 weeks of incubation at 37°C, MAP colonies were processed for isolation, identification and molecular characterization to check the purity of culture. Afterwards used for spiking in feces at different dilutions.
 
Procurement of MAP specific LAMP primers using IS900 gene
 
A total of six HPLC purified MAP specific primers including 2 outer primers (F3 and B3), 2 inner primers (FIP and BIP) and 2 loop primers (LF and LB) were commercially procured from ILS, USA. These primers recognized eight distinct regions on the target MAP DNA and were designed by Heidarnejhad et al., (2015).
 
IS900 PCR
 
MAP colonies were characterized by IS900 PCR as per Singh et al., (2010) using P90 and P91 primers (Millar et al., 1995). Colonies (2 loopful) were harvested in sterilized normal saline and pelleted after centrifugation at 12,000 RPM at RT for 15 min. Then DNA isolation was done as stated by van Embden et al., (1993) with minor modifications. In brief, 12.5 μl of 2X green dye PCR master mix (Promega), 1 μl forward primer (10 pmole/μl) and 1μl reverse primer (10 pmole/μl), 7.5μl of nuclease free water and 3μl of template MAP DNA was added to a 0.2 mL PCR tube (total volume 25 μl). Thermal cycling conditions were set as: initial denaturation at 94°C for 3 min, followed by 35 cycles including denaturation at 94°C for 30 sec, annealing at 64°C for 30sec, extension at 72°C for 1 min and final extension step at 72°C for 7 min. Amplicon produced of 413 bp sizes were taken as positive, after separating on 2% agarose gel stained with ethidium bromide.
 
Optimization of reaction conditions for LAMP
 
LAMP test was carried out in tubes containing 25μl of final reaction mixture containing 10× Thermopol® Reaction Buffer (New England Biolabs, USA), MAP specific LAMP primers (F3, B3, FIP, BIP, FLP and BLP), MgSO4 (New England Biolabs, USA), dNTP mix (New England Biolabs, USA), Betaine (Sigma Aldrich), 8 U Bst DNA polymerase 2 (New England Biolabs, USA), calcein dye (Sigma Aldrich), MnCl2, KCl, Triton X-100, nuclease free water and MAP DNA template. Based on the previous studies (Heidarnejhad et al., 2015; Safi et al., 2015), different combinations of a range of concentrations of each constituent (dNTPs (0.4 mmol/L ~1.6 mmol/L), betaine (0.8 mmol/L ~1.4 mmol/L), MgSO4 (2 mmol/L ~9 mmol/ L) were examined for amplification efficiency. The amplification reaction was carried out in a thermal cycler between 59°C to 65°C within 30 to 80 min, to determine the optimal incubation temperature and time. At the end of each incubation step, the reaction was stopped by heating at 95°C for 2 min. All of the experimentations were repeated four times.
 
Analysis of MAP specific LAMP products
 
Before amplification, visual examination of the MAP specific LAMP assay products was evaluated by adding fluorescence components (calcein and MnCl2; Sigma Aldrich) to the reaction mixture and a color change in the reaction mixture was recorded after successful amplification. Samples turned into green color were considered as positive, while samples remained in orange color were taken as negative. On the other hand, 10 µL of the MAP specific LAMP amplicons were examined by 1.8% agarose gel electrophoresis. The presence of a smear or 5multiple bands pattern with different molecular weights confirmed the positive results (Fig 3).
 
Optimization of LAMP from genomic DNA of MAP culture (106 CFU to 1 CFU)
 
Genomic DNA was isolated as per Van Embden et al., (1993) with minor modifications. Sensitivity of the LAMP assay on MAP culture was optimized by taking 6-fold serial dilutions made from 431 ng/μl MAP stock solution in 1mLof nuclease free water. A series of ten-fold dilutions (1 × 108 to 1 × 10° copies/mL) were used to evaluate the sensitivity of the MAP specific LAMP assay.
 
Detection of clinical samples by MAP specific LAMP assay
 
A total of 221 fecal samples (85 goats, 47 sheep, 54 cows and 35 buffaloes) were collected from farm goatherds located at Central Institute for Research on Goats (CIRG), Makhdoom and places around Agra and Mathura districts of Uttar Pradesh, North India. MAP DNA isolation protocol was standardised in our laboratory. Briefly, fecal sample (0.1 g) was homogenized in 500 µL stool lysis buffer (Qiagen), settled down at room temperature for 25 min and then supernatant was filtered using 0.22µm pore size syringe filters (Sigma Aldrich). The filtrates containing extracted DNAs were checked by both the MAP specific LAMP assay and traditional IS900 PCR.
 
Statistical analysis
 
Statistical significance was measured between two tests, Mc Nemar’s test and kappa agreement statistical analysis methods applied by Graph Pad software, USA. Limit of detection (LOD), sensitivity and specificity of LAMP PCR has been calculated using online MedCalc software (https://www.medcalc.org/).

RESULTS AND DISCUSSION

MAP specific LAMP assay
 
MAP specific LAMP assay was carried out in a 25 µL reaction mixture after optimizing the reaction conditions. Final reaction mixture contained 2.5 µL 10× Thermopol® Reaction Buffer (20 mM Tris–HCl, 10 mM (NH4)2SO4, 10mMKCl, 2mMMgSO4, 0.1% Triton X-100), 60 pM each of F3, B3, FIP and BIP, 30 pM each of FLP and BLP, 7.5 mM MgSO4, 40 mM dNTPs, 0.8 M Betaine (Sigma Aldrich, USA), 8U Bst DNA polymerase (New England Biolabs, USA), 2.5 mmol/L calcein, 50 mmol/L MnCl2, 2.5µL template MAP DNA and nuclease free water to make the final volume up to 25 µL. Prior to amplification, the initial color of the reaction solution was orange. The optimal reaction time and incubation temperature were set at 62°C for 30 min. The LAMP reaction was performed in a waterbath (Remi). For comparison purpose, it was also performed n a conventional thermal cycler. Detection limit of the LAMP was defined at specific dilution conditions and the reactions were performed four times to evaluate the reproducibility of the LAMP assay.
 
Specificity and sensitivity of MAP specific LAMP assay
 
The standardized MAP specific LAMP assay was also used to specifically amplify two other Mycobacterial species viz. Mycobacterium tuberculosis and Mycobacterium avium. Test results (Fig 1A and B) illustrated that this LAMP technique does not exhibited any cross-reactivity with other tested pathogenic Mycobacterial species. Two other Mycobacterial species were also examined by the MAP specific LAMP assay resulting in the presence of positive green color in all final products of the LAMP assay (Fig 1B, lanes 4 to 6) and a typical DNA ladder pattern was showed after separating in 1.5% agarose gel electrophoresis (Fig 1A, lanes 4 to 6). The amplicons of the MAP specific LAMP assay for other pathogenic Mycobacterial species remained negative with orange color (no color change) (Fig 1B, lanes 2 and 3); additionally, the other pathogenic Mycobacterial species lacked the typical DNA ladder pattern, emphasizing on the absence of amplification (Fig 1A, lanes 2 and 3).
 

Fig 1: Specificity of MAP specific LAMP assay:


       
The sensitivity of the MAP specific LAMP assay was then evaluated and compared with that of traditional IS900 PCR. The detection limit of the MAP specific LAMP assay is evaluated upto ten copies (Fig 2A and B). But, the resulted detection limit of traditional IS900 PCR was 1 × 103 copies (Fig 3). This specifies that the sensitivity of the MAP specific LAMP assay was 100-fold higher than that of traditional IS900 PCR. Our results exhibited that the MAP specific LAMP assay is highly specific, sensitive and superior to the traditional IS900 PCR to detect MAP infection.
 

Fig 2: Sensitivity of the MAP specific LAMP assay.


 

Fig 3: Sensitivity of traditional IS900 PCR.


 
Detection of clinical samples by MAP specific LAMP assay: The MAP specific LAMP assay was used to test 221 fecal samples of domestic livestock species, which were also tested by microscopy, conventional IS900 PCR and culture for comparison with the MAP specific LAMP assay. Of 221 samples (Goats- 85, Sheep- 47, Cows- 54 and Buffaloes- 35) screened, 29.1 (65), 32.1 (71), 21.7 (48) and 23.5% (52) were positive for presence of MAP in LAMP assay, microscopy, IS900 PCR and culture, respectively (Table 1). Except microscopy, The MAP specific LAMP assay had 100% of agreement (minimum number of positive samples) with IS900 PCR and culture in testing these fecal samples. However, MAP specific LAMP assay was noted to be a quicker, easier and more cost-efficient method compared to other tests.
 

Table 1: Screening of fecal samples from farm goatherds located at CIRG, Makhdoom and places around Agra and Mathura districts of Uttar Pradesh for the presence of MAP using multiple tests.


       
Statistically kappa and two-tailed p values were calculated for the four tests used for the screening of 221 fecal samples (Table 2). All four tests were compared statistically and the MAP specific LAMP assay had almost perfect agreement having kappa value of 0.914 with respect to microscopy, substantial agreement having kappa value of 0.759 with respect to IS900 PCR and almost perfect agreement having kappa value of 0.824 with respect to culture, respectively. With respect to microscopy, IS900 PCR and culture, MAP specific LAMP assay had sensitivity of 98.6, 93.7 and 96.8%, respectively. Specificity of MAP specific LAMP assay with respect to microscopy, IS900 PCR and culture was 96.1, 92.2 and 92.3%, respectively (Table 2).
 

Table 2: Sensitivity and specificity of visual LAMP assay vis a vis diagnostic tests for the screening of feces of Domestic livestock species (n= 221).


       
Johne’s disease (JD) or paratuberculosis is an incurable chronic inflammation of the intestine that affects ruminant species is global in distribution and have variable prevalence 5-55.0% (clinically) (Giese and Ahrens, 2000) and 2-12.0% (sub-clinically) (Jakobsen et al., 2000). In India high bio-load of MAP (25.0 to 60.0%) was reported in farm animals (Singh et al., 2014a). There are concerns that MAP may play role in human autoimmune diseases like inflammatory bowel disease (Crohn’s disease), type I diabetes, etc. (Sechi et al., 2015; Chaubey et al., 2017). RNA of MAP has also been identified in intestinal tissues of Crohn’s disease patients (Greenstein, 2003).
       
Fecal microscopy is simple, fast and inexpensive, however is low sensitive (49%) and specific (83%) (Zimmer et al., 1999). Johnin PPD based DTH testing (intradermal skin testing) is another popular simple field based test, however estimated sensitivity (54.0%) and specificity (79.0%) is low (Kalis et al., 2003). Fecal culture though ‘Gold standard’, however is poorly sensitive (15-20%) due to long incubation period, use of harsh decontamination procedures, low bacilli load in early stages, intermittent shedding etc (Tiwari et al., 2006). Fecal PCR takes less time, however again sensitivity is low (23%) due to factors like inhibitory substances, low load of MAP in early stages, intermittent shedding, inefficient DNA isolation methods (Gilardoni et al., 2009). Commercially available ELISAs are based on whole cell antigens, therefore suffers with disadvantage of poor specificity due to cross reactive antigens with other mycobacteria (Gupta et al., 2016). Moreover, estimated sensitivity of commercial ELISAs is very poor (13.5 to 42%) (Singh et al., 2007). Hence, to diagnose and control JD, there is a need to develop a rapid and efficacious diagnostics for JD so that control of disease is initiated.
       
In recent years, use of LAMP assay for diagnosis of both human and animal diseases is increasing. LAMP is widely being studied for detecting infectious diseases such as tuberculosis (Dhanasekaran et al., 2011), sleeping sickness (Nijru et al., 2008), foot and mouth disease (Dukes et al., 2006). This assay is routinely used for specific and rapid detection of Brucella abortus (Karthik et al., 2014), Babesia felis (Salim et al., 2018), Marek’s disease of chickens (Woznrakowski and Salamonowicz, 2014), visceral leishmaniasis (Khan et al., 2012) etc. In addition to above developments, LAMP has also been developed for diagnosis of many human diseases like human papilloma virus (Hagiwara et al., 2007), Human herpex virus (Ihira et al., 2007), HIV-1 (Cutis et al., 2008), West Nile virus (Parida et al., 2004), H1N1 virus (Kubo et al., 2010) etc.
       
The primary advantage of LAMP in comparison with traditional IS900 PCR is that the result of amplification can be evaluated without post amplification processing (no need of agarose gel electrophoresis or instruments like UV trans-illuminator or gel-doc system etc). Visual turbidity signifies the positive reaction and proved to be one of the good indicators (Dhama et al., 2014). The independence from the use of thermal cycler in LAMP makes the assay suitable for the detection of MAP in low facility laboratory setups (Sahoo et al., 2016). In LAMP test, amplification of nucleic acids is carried out under isothermal conditions requiring less time compared to traditional PCR without affecting the sensitivity and specificity of test (Nirju et al., 2012). LAMP has high tolerance to inhibitory substances from culture medium or biological substances which influences the efficiency of traditional IS900 PCR (Kaneko et al., 2007). It is cheaper as compared to real time PCR (Yamazaki et al., 2013). Sensitivity of LAMP is higher than that of conventional PCR and non–denatured template can be used for amplification (Nagamine et al., 2001).
       
Lately, there was an interest in developing LAMP test for JD. Gene sequences like IS900 (Safi et al., 2015), HSPX (Enosava et al., 2003) and F57 (Enosava et al., 2003) have been used to develop LAMP primers. Firstly, Enosava et al., (2003) optimized LAMP test on bacterial culture (20 different MAP strains) for MAP using IS900, HspX and F57. This test took upto 3 hours as it didn’t have loop primers specific for MAP. However, there are no Indian reports available on use of MAP specific LAMP PCR for the detection of JD. This present study is the first report which utilized the efficiency of MAP specific LAMP assay and also standardized using MAP ‘S 5’ strain of ‘Indian Bison Type’ bio-type which is endemic (83.3%) in domestic livestock of the country (Singh et al., 2014a).
       
In contrast, the present study utilized the specific loop primers for amplifying MAP DNA of 221 fecal samples of domestic livestock using LAMP PCR showing visible results in thirty minutes without any use of gel electrophoresis. LAMP PCR results in present study revealed that the visible results were observed upto the detection limit of 10 femtograms of MAP DNA present in fecal sample, which was 100 times greater than that of Traditional IS900 PCR.
       
Recently workers like Heidarnejhad et al., (2015) and Safi et al., (2015) have also optimized LAMP assay on bacterial culture (wild-type MAP isolate) and clinical fecal samples using IS900 for designing of loop primers. This reduced the processing timings upto 2 hours. The resulted sensitivity was 100 times greater than that of traditional and Nested-PCR. However, these studies didn’t use unprocessed or partially processed samples for diagnostics and faced some limitations viz., long incubation time (2 hours). But in the present study, we used partially processed fecal samples (n- 221) direct as a sample or template for LAMP PCR to detect MAP. Of 221 fecal samples, results revealed that the sensitivity and specificity of MAP specific LAMP PCR was found to be 100% and 95.7% when compared to the traditional IS900 PCR. Sensitivity of LAMP PCR was 98.6% and 96.1% when compared to microscopy and culture, respectively. Whereas, LAMP PCR found 96.1% and 92.3% specific after comparing with microscopy and culture, respectively (Table 2). Statistical comparisons were evaluated and the strength of agreement for LAMP PCR was estimated to be ‘Almost perfect’ with kappa values of 0.914, 0.859 and 0.824 with respect to microscopy, traditional IS900 PCR and culture, respectively (Table 2).
       
Since, fecal samples have been regarded as the main source of transmission of MAP from infected animals to healthy animals. In present study, we evaluated the ability of MAP specific LAMP assay to detect MAP bacilli in fecal samples of herd animals, since fecal samples were easy to collect and didn’t require any expertise in collection of samples. In the present study we compared the diagnostic potential of MAP specific LAMP assay vis a vis microscopy using ZN staining, IS900 PCR and culture for the detection of MAP in fecal samples. MAP specific LAMP assay was proved to be a simple, fast, highly sensitive and specific procedure for the detection of MAP bacilli in fecal samples. Since, the economy of dairy animals is linked to trading requires a sanitary control program with the purpose of eradicating the disease, therefore we evaluated its ability in the early detection of MAP bacilli in fecal samples specifically.

CONCLUSION

MAP specific LAMP assay was found to be highly sensitive and specific for the detection of MAP in fecal samples of domestic livestock. Unlike ZN staining which detects the presence of Mycobacterium, MAP specific LAMP assay was specific and accurate so far the detection of the presence of MAP bacilli in fecal samples. Based on the above findings, we conclude that the visual LAMP assay is user friendly, cost effective rapid, simple and can performed by non-skilled persons in low facility laboratory settings where resources are limited mainly in developing countries like India.However, repetition of mis-match samples was more prudent way to resolve and involve few more tests on these samples.

ACKNOWLEDGEMENT

Authors are thankful to Directors (Central Institute for Research on Goats, Makhdoom and AIMT, AMITY University, Jaipur) for providing laboratory facilities.

CONFLICT OF INTEREST

No conflict of Interest to declare.

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